The long-range objective of this proposal is to understand the structure- function relationship of the ubiquitous ankyrin-like (ANK) repeat motif. The ANK repeat is a 33-amino acid motif, which is found in tandem arrays of multiple copies in a wide range of proteins. Originally discovered in the cell-cycle regulators CDC10/SW16 and the Drosophila membrane receptor Notch, the ANK motif has been identified in hundreds of proteins in nearly all phyla. A growing body of evidence has shown that the ANK motif mediates diverse protein-human diseases. Recent structural studies from our laboratory and other have revealed that despite the loose consensus sequence, ANK repeats in different ANK repeat-bearing proteins adopt a characteristic L shape comprising a protruding hairpin-like tip followed bu a helix-turn-helix motif, and the multiple repeat copies stack sequentially to form elongated structures. While these initial studies have provided insights into the structural features of the ANK repeat, the mechanism of how the motif utilizes the conserved fold and multiple copies to confer specificity in diverse molecular processes still remains puzzling. Continued effort is clearly needed to further characterize and understand this ubiquitous and multi-functional motif. In this study, we propose to focus on a thorough structural, dynamic, and biochemical analysis of IkappaBalpha, a functionally well-known ANK repeat-bearing protein. IkappaBalpha represents a major subclass of the ANK family: IkappaB proteins, which inhabit the ubiquitous transcription factor NFkappaB and regulate the expression of many cellular genes involved in stress and immune response. This work will not only provide a detailed molecular insight into how IkappaBalpha binds to and inhibits NFkappaB but also contribute significantly to the understanding of the structural basis of the multi-functional ANK repeat motif. The specific Aims 1 and 2 will determine the solution structures and dynamics of the IkappaBalpha inhibitory domain containing 6 ANK repeats (23 kDA) and its complex (25 kDa) with the target peptide from NFkappaB by multi-dimensional heteronuclear NMR spectroscopy; Aim 3 is to examine the contributions of individual residue in the ANK motif involved in binding to NFkappaB and to evaluate the role of multiple ANK copies in the IkappaB structure and function by mutagenesis and other biochemical/biophysical approaches. The information derived from this work may be useful for the understanding and treatment of stress and immune-related diseases, which involve the malfunctions of IkappaB-NFkappaB signaling pathway.